Screening Of High Chitinolytic Bacillus,Cloning And Expression Of Chitinase Gene

Six spore-forming chitinolytic bacteria were isolated from soil of Wuhan in current study.The 16 S rRNA gene sequencing demonstrated that four strains(D1,D2,D3 and D4)can be classified as Paenibacillus illinoisensis.Two of them(CDY-607 and W1)phylogenetically related to Paenibacillus xylanilyticus.Strain CDY-607 which has a Higher chitinolytic activity was selected for the further study.The results are listed as follows:The optimum temperature for chitinase generation was 55?,the optimum pH for enzymatic activity were 6.5 and 8.0;Fe2+ had promoted the enzymatic activity in fermentation culture.The fermentation medium and culture conditions were optimized by single factor and orthogonal experimental design.The results shows that the optimum fermentation time for chitinase generation was 96 h,the optimum inoculation amount was 5%,the optimum culture volume was 100 mL,and the optimum medium pH was 6.5.The optimum media components were colloidal chitin 5 g/L,FeSO4 2 g/L,peptone 2 g/L and yeast extract 5 g/L.After optimization,the enzymatic activity in the fermentation culture achieved 49.26 mU/m L,which was two times of the enzymatic activity(24.62 mU/m L)detected in former conditions.The sequences on both sides of conserved DNA fragment(426 bp)were amplified by Tail-PCR.The coting sequence revealed that the ORF of this chitinase is 996 bp.This ORF encoded only one domain,the 18 th glucosidase family chitinase catalytic domain,which is 912 bp and constituted by 304 amino acids,and its prediction molecular weight is 33.42 kDa.The chitinase gene was cloned and ligated into vector pFLAG-CTS,and this chitinase was successfully secreted by E.coli DH5 a.After induced with IPTG for 12 h,the chtinolytic activity of fermentation culture was 17 mU/m L.SDS-PAGE also detected the secretion of this chitinase,and its molecular weight was about 33.4 kDa,which was consistent with the predicted result.